The long term goal of this proposal is to identify novel mechanisms involved in melanoma initiation and maintenance with the ultimate objective of a more rational approach to treatment. Nearly all individuals of fair skin bear multiple benign melanocytic neoplasms (nevi) expressing pro- oncogenic mutations in the MAPK pathway, usually in the BRAF or NRAS genes. Still, these mutated nevus cells can remain mitotically inactive for decades. The molecular bases blocking the malignant transformation of nevi into melanoma are poorly defined. We have previously reported that cellular senescence may be a potent mechanism accounting for the silent nature of melanocytes expressing oncogenic BRAF, both in vitro and in vivo. However, the size and histological features of nevi are notoriously heterogeneous. Therefore, it is unlikely that a single suppressive response controls early stages of melanocyte transformation. We have recently found that tumor-associated BRAF and RAS proteins are opposed by two kinetically, morphologically and mechanistically different anti-proliferative responses. BRAFV600E induced a slow program of senescence that could be rescued by additional oncogenes. In contrast, NRASQ61R and particularly HRASG12V, engaged a fast cell cycle arrest associated with massive disorganization of the cellular architecture and the activation of endoplasmic reticulum (ER)-associated stress (specifically, the Unfolded Protein Response, UPR). In this proposal we will test the hypothesis that the ER is a key rheostat of oncogene and drug-induced stress signals, and as such it plays a critical role in melanoma initiation, progression and chemoresistance. To test this hypothesis, we will address the molecular bases underlying the induction of melanocyte senescence by RAS proteins (Aim 1). Special emphasis will be dedicated to reconstructed human skin, genetically modified to selectively target ER-modulators and determine their impact on melanocytic neoplasia in a three dimensional model (Aim 2). In parallel, a combination of fluorescence-based and nuclear magnetic resonance imaging will be used to dissect the functional contribution of the UPR to melanoma maintenance and chemoresistance in animal models (Aim 3). The identification and characterization of novel tumor suppressor mechanism(s) in melanoma is likely to have important clinical implications. In addition, since altered stress responses are a general feature of multiple tumor types, the proposed studies should be relevant to a wide variety of other aggressive cancers, and may provide a new class of targets for drug development. PUBLIC HEALTH RELEVANCE: Melanoma is the most aggressive form of skin cancer. The long-term survival for patients with Stage IV (metastatic) melanoma is typically limited to 6-10 months, and has not improved since the 1970s130,131. Therefore, the melanoma field is in urgent need of new markers of the disease and improved anticancer strategies. The primary objective of this project is to evaluate the contribution of survival signals dependent on the endoplasmic reticulum to the aggressive behavior of melanoma cells and their extreme chemoresistance. We consider that one of the most innovative aspects of this proposal is the engineering of melanocytic lesions with specific genetic defects, and subsequent functional studies in vitro and in vivo beyond standard tissue culture systems. In addition, a close collaboration with one of the largest Multidisciplinary Melanoma Clinics of the country will help testing and validating the physiological impact of our hypotheses. We expect that our studies will provide new knowledge of the molecular basis of melanoma progression, and reveal novel targets for therapeutic intervention.